This invention relates, in general, to insoluble reagents useful in performing immunoassays for hepatitis, and more particularly to a water insoluble polymeric disc having a layer of antibody reactive with antigens associated with hepatitis covalently bonded to a surface which is designed to increase the contact between the antibody and the antigen.
Insoluble solids used to immobilize water soluble biological substances such as proteins, enzymes, antigens, and antibodies are known. Examples of such solids may be found in U.S. Pat. No. 3,853,987 to W. J. Dreyer, U.S. Pat. No. 3,666,733 to R. Epton, and U.S. Pat. No. 3,645,852 to R. E. A. Axen et al. One use for such solids, when bonded to a suitable material, is in the detection of antigens or antibodies associated with diseases found in human blood.
Since the recent discovery of antigens associated with hepatitis, a number of procedures have been developed which are designed to detect the presence of the antigen in blood by utilizing insolubilized reagents and the well-known antigen-antibody reaction. One such process is disclosed in U.S. Pat. No. 3,867,517 entitled Direct Radioimmunoassay for Antigens and their Antibodies, by Chung-Mei Ling. This patent discloses a method for direct immunoassay of antigens which utilizes a coated test well, i.e., an insoluble solid, onto which the hepatitis antibody is adsorbed.
Another process for detecting hepatitis antigen is disclosed in copending U.S. patent application Ser. No. 617,743, entitled Method of Determining the Presence of an Antigen Associated with Hepatitis, by Seymour P. Halbert et al, filed Sept. 29, 1975, the teachings of which are incorporated herein by reference. That application discloses a sensitive, rapid technique for detecting antigens associated with hepatitis and present in body fluids. Briefly, the technique involves incubating a small sample of body fluid to be tested for antigens associated with hepatitis with an insoluble, disc-like solid matrix having an antibody reactive with hepatitis antigens bonded to its surface. If the sample contains an antigen associated with hepatitis, a bond forms between the immobilized antibody and the antigen found in the test sample. The reagent on the solid matrix is then incubated with hepatitis antibody, tagged or labeled with an enzyme capable of catalyzing a reaction of a substrate to form a detectable end product. If a hepatitis antigen was present in the test sample, a "sandwich" structure is formed comprising the insoluble matrix, hepatitis antibody, hepatitis antigen, and enzyme tagged hepatitis antibody. A third incubation, this time with a solution of suitable enzyme substrate, will indicate the presence of the antigen in the test sample since the now immobilized enzyme will catalyze the formation of an end product measurable with a photometric detector.
The ideal test for a hepatitis antigen should be easily performed, highly sensitive, rapid, capable of eliminating false positives, utilizing stable reagents and requiring only a very small sample of test body fluid. The reagents used in any hepatitis detection system should be designed as much as possible with a view of these ends.
Accordingly, it is an object of the present invention to provide an insoluble matrix useful as described in the abovementioned tests which has a high concentration of purified, hepatitis antibody distributed uniformly over its entire surface.
A further object is to attach hepatitis antibody to a solid such that it will not be dislodged by mechanical or chemical forces to which it may be subjected during use. In this regard, it is imperative to effect the attachment such that the antibody remains functional, i.e., capable of participating in its immunological reaction and immunochemically unaltered by its attachment.
A further object of the invention is to provide an improved shape for an insoluble antibody coated solid which is easy to handle during immunoassay and which is designed to expose a greater portion of its coated surface to the solutions used during incubations.